Clinical Applications of Hemolytic Markers in the Differential Diagnosis and Management of Hemolytic Anemia

Several hemolytic markers are available to guide the differential diagnosis and to monitor treatment of hemolytic conditions. They include increased reticulocytes, an indicator of marrow compensatory response, elevated lactate dehydrogenase, a marker of intravascular hemolysis, reduced haptoglobin, and unconjugated hyperbilirubinemia. The direct antiglobulin test is the cornerstone of autoimmune forms, and blood smear examination is fundamental in the diagnosis of congenital membrane defects and thrombotic microangiopathies. Marked increase of lactate dehydrogenase and hemosiderinuria are typical of intravascular hemolysis, as observed in paroxysmal nocturnal hemoglobinuria, and hyperferritinemia is associated with chronic hemolysis. Prosthetic valve replacement and stenting are also associated with intravascular and chronic hemolysis. Compensatory reticulocytosis may be inadequate/absent in case of marrow involvement, iron/vitamin deficiency, infections, or autoimmune reaction against bone marrow-precursors. Reticulocytopenia occurs in 20-40% of autoimmune hemolytic anemia cases and is a poor prognostic factor. Increased reticulocytes, lactate dehydrogenase, and bilirubin, as well as reduced haptoglobin, are observed in conditions other than hemolysis that may confound the clinical picture. Hemoglobin defines the clinical severity of hemolysis, and thrombocytopenia suggests a possible thrombotic microangiopathy or Evans' syndrome. A comprehensive clinical and laboratory evaluation is advisable for a correct diagnostic and therapeutic workup of the different hemolytic conditions

Autoimmune Cytopenias in Chronic Lymphocytic Leukemia: Focus on Molecular Aspects

Autoimmune cytopenias, particularly autoimmune hemolytic anemia (AIHA) and immune thrombocytopenia (ITP), complicate up to 25% of chronic lymphocytic leukemia (CLL) cases. Their occurrence correlates with a more aggressive disease with unmutated VHIG status and unfavorable cytogenetics (17p and 11q deletions). CLL lymphocytes are thought to be responsible of a number of pathogenic mechanisms, including aberrant antigen presentation and cytokine production. Moreover, pathogenic B-cell lymphocytes may induce T-cell subsets imbalance that favors the emergence of autoreactive B-cells producing anti-red blood cells and anti-platelets autoantibodies. In the last 15 years, molecular insights into the pathogenesis of both primary and secondary AIHA/ITP has shown that autoreactive B-cells often display stereotyped B-cell receptor and that the autoantibodies themselves have restricted phenotypes. Moreover, a skewed T-cell repertoire and clonal T cells (mainly CD8+) may be present. In addition, an imbalance of T regulatory-/T helper 17-cells ratio has been involved in AIHA and ITP development, and correlates with various cytokine genes polymorphisms. Finally, altered miRNA and lnRNA profiles have been found in autoimmune cytopenias and seem to correlate with disease phase. Genomic studies are limited in these forms, except for recurrent mutations of KMT2D and CARD11 in cold agglutinin disease, which is considered a clonal B-cell lymphoproliferative disorder resulting in AIHA. In this manuscript, we review the most recent literature on AIHA and ITP secondary to CLL, focusing on available molecular evidences of pathogenic, clinical, and prognostic relevance

The Changing Landscape of Autoimmune Hemolytic Anemia

Autoimmune hemolytic anemia (AIHA) is a greatly heterogeneous disease due to autoantibodies directed against erythrocytes, with or without complement activation. The clinical picture ranges from mild/compensated to life-threatening anemia, depending on the antibody's thermal amplitude, isotype and ability to fix complement, as well as on bone marrow compensation. Since few years ago, steroids, immunesuppressants and splenectomy have been the mainstay of treatment. More recently, several target therapies are increasingly used in the clinical practice or are under development in clinical trials. This has led to the accumulation of refractory/relapsed cases that often represent a clinical challenge. Moreover, the availability of several drugs acting on the different pathophysiologic mechanisms of the disease pinpoints the need to harness therapy. In particular, it is advisable to define the best choice, sequence and/or combination of drugs during the different phases of the disease. In particular relapsed/refractory cases may resemble pre-myelodysplastic or bone marrow failure syndromes, suggesting a careful use of immunosuppressants, and vice versa advising bone marrow immunomodulating/stimulating agents. A peculiar setting is AIHA after autologous and allogeneic hematopoietic stem cell transplantation, which is increasingly reported. These cases are generally severe and refractory to standard therapy, and have high mortality. AIHAs may be primary/idiopathic or secondary to infections, autoimmune diseases, malignancies, particularly lymphoproliferative disorders, and drugs, further complicating their clinical picture and management. Regarding new drugs, the false positivity of the Coombs test (direct antiglobulin test, DAT) following daratumumab adds to the list of difficult diagnosis, together with the passenger lymphocyte syndrome after solid organ transplants. Diagnosis of DAT-negative AIHAs and evaluation of disease-related risk factors for relapse and mortality, notwithstanding improvement in diagnostic approach, are still an unmet need. Finally, AIHA is increasingly described following therapy of solid cancers with inhibitors of immune checkpoint molecules. On the whole, the double-edged sword of new pathogenetic insights and therapies has changed the landscape of AIHA, both providing enthusiastic knowledge and complicating the clinical management of this disease

Mesenchymal Stem Cells in Aplastic Anemia and Myelodysplastic Syndromes: The "Seed and Soil" Crosstalk

There is growing interest in the contribution of the marrow niche to the pathogenesis of bone marrow failure syndromes, i.e., aplastic anemia (AA) and myelodysplastic syndromes (MDSs). In particular, mesenchymal stem cells (MSCs) are multipotent cells that contribute to the organization and function of the hematopoietic niche through their repopulating and supporting abilities, as well as immunomodulatory properties. The latter are of great interest in MDSs and, particularly, AA, where an immune attack against hematopoietic stem cells is the key pathogenic player. We, therefore, conducted Medline research, including all available evidence from the last 10 years concerning the role of MSCs in these two diseases. The data presented show that MSCs display morphologic, functional, and genetic alterations in AA and MDSs and contribute to immune imbalance, ineffective hematopoiesis, and leukemic evolution. Importantly, adoptive MSC infusion from healthy donors can be exploited to heal the "sick" niche, with even better outcomes if cotransplanted with allogeneic hematopoietic stem cells. Finally, future studies on MSCs and the whole microenvironment will further elucidate AA and MDS pathogenesis and possibly improve treatment

Management of refractory autoimmune hemolytic anemia after allogeneic hematopoietic stem cell transplantation : current perspectives

Autoimmune hemolytic anemia (AIHA) is increasingly observed after allogeneic hematopoietic stem cell transplantation (allo-HSCT), with a reported incidence between 4% and 6%. The disease is generally severe and refractory to standard therapy, with high mortality, and there are neither defined therapies, nor prospective clinical trials addressing the best treatment. Most of the knowledge on the therapy of AIHAs derives from primary forms, which are highly heterogeneous as well, further complicating the management of post-allo-HSCT forms. The review addresses the risk factors associated with post-allo-AIHA, including unrelated donor, the development of chronic extensive graft-versus-host disease, CMV reactivation, nonmalignant diagnosis pre-HSCT, and alemtuzumab use in conditioning regimens. Regarding therapy, we describe standard treatments, such as corticosteroids, intravenous immunoglobulin, splenectomy, rituximab, cyclophosphamide, and plasma exchange, which have lower response rates than those reported in primary forms. New therapeutic options, including sirolimus, bortezomib, abatacept, daratumumab and complement inhibitors, are promising tools for this detrimental complication occurring after allo-HSCT

Small Paroxysmal Nocturnal Hemoglobinuria Clones in Autoimmune Hemolytic Anemia: Clinical Implications and Different Cytokine Patterns in Positive and Negative Patients

Autoimmune hemolytic anemia (AIHA) is characterized by immune mediated erythrocytes destruction by autoantibodies with or without complement activation. Additional pathologic mechanisms include cellular cytotoxicity, cytokline dysregulation, and inadequate bone marrow compensation with fibrosis/dyserythropoiesis. The latter resembles that of bone marrow failures, namely aplastic anemia and myelodysplastic syndromes. Paroxysmal nocturnal hemoglobinuria (PNH) clones are increasingly recognized in bone marrow failure syndromes, and their selection and expansion are thought to be mediated by immune mechanisms. In this study, we aimed to evaluate the prevalence of PNH clones in 99 patients with primary AIHA, and their correlations with disease features and outcomes. Moreover, in the attempt to disclose the physiopathology of PNH positivity in AIHA, serum levels of several immunomodulatory cytokines were tested. A PNH clone was found in 37 AIHA patients (37,4%), with a median size of 0.2% on granulocytes (range 0.03\u201385). Two patients showed a large clone (16 and 85%) and were therefore considered as AIHA/PNH association and not included in further analysis. Compared to PNH negative, PNH positive cases displayed a higher hemolytic pattern with adequate bone marrow compensation. AIHA type, response to therapy, complications and outcome were comparable between the two groups. Regarding cytokine levels, IFN-\u3b3 and IL-17 were lower in PNH positive vs. PNH negative AIHAs (0.3 \ub1 0.2 vs. 1.33 \ub1 2.5; 0.15 \ub1 0.3 vs. 3,7 \ub1 9.1, respectively, p = 0.07 for both). In PNH positive AIHAs, IFN-\u3b3 positively correlated with reticulocytes (r = 0.52, p = 0.01) and with the bone marrow responsiveness index (r = 0.69, p = 0.002). Conversely, IL-6 and IL-10 showed the same pattern in PNH positive and PNH negative AIHAs. IL-6 levels and TGF-\u3b2 positively correlated with clone size (r = 0.35, p = 0.007, and r = 0.38, p = 0.05, respectively), as well as with LDH values (r = 0.69, p = 0.0003, and r = 0.34, p = 0.07, respectively). These data suggest testing PNH clones in AIHA since their prevalence is not negligible, and may correlate with a prominent hemolytic pattern, a higher thrombotic risk, and a different therapy indication. PNH testing is particularly advisable in complex cases with inadequate response to AIHA-specific therapy. Cytokine patterns of PNH positive and negative AIHAs may give hints about the pathogenesis of highly hemolytic AIHA

Parallel processing in immune networks

In this work we adopt a statistical mechanics approach to investigate basic, systemic features exhibited by adaptive immune systems. The lymphocyte network made by B-cells and T-cells is modeled by a bipartite spin-glass, where, following biological prescriptions, links connecting B-cells and T-cells are sparse. Interestingly, the dilution performed on links is shown to make the system able to orchestrate parallel strategies to fight several pathogens at the same time; this multitasking capability constitutes a remarkable, key property of immune systems as multiple antigens are always present within the host. We also define the stochastic process ruling the temporal evolution of lymphocyte activity, and show its relaxation toward an equilibrium measure allowing statistical mechanics investigations. Analytical results are compared with Monte Carlo simulations and signal-to-noise outcomes showing overall excellent agreement. Finally, within our model, a rationale for the experimentally well-evidenced correlation between lymphocytosis and autoimmunity is achieved; this sheds further light on the systemic features exhibited by immune networks.Comment: 21 pages, 9 figures; to appear in Phys. Rev.

Autoimmune cytopenias in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is frequently complicated by secondary autoimmune cytopenias (AIC) represented by autoimmune hemolytic anemia (AIHA), immune thrombocytopenia (ITP), pure red cell aplasia and autoimmune granulocytopenia. The distinction of immune cytopenias from cytopenias due to bone marrow infiltration, usually associated with a worse outcome and often requiring a different treatment, is mandatory. AIHA and ITP are more frequently found in patients with unfavorable biological risk factors for CLL. AIC secondary to CLL respond less favorably to standard treatments than their primary forms, and treating the underlying CLL with chemotherapy or monoclonal antibodies may ultimately be necessary

Biological and molecular characterization of PNH-like lymphocytes emerging after Campath-1H therapy

Campath-1H, an anti-CD52 monoclonal antibody, is therapeutically active in lymphoproliferative and autoimmune diseases. After Campath-1H therapy, lymphocytes with a paroxysmal nocturnal haemoglobinuria (PNH) phenotype have been reported to emerge. We characterized a PNH-like lymphocyte population emerging after Campath-1H therapy, in a patient with fludarabine refractory B-cell chronic lymphocytic leukaemia (B-CLL). We demonstrated a reduction in PIG-A mRNA levels compared with controls, and of all cytokines tested [interleukin (IL)-4, IL-13, IL-2, interferon(IFN)-gamma, IL-6, IL-10, and tumour necrosis factor (TNF)-alpha], except transforming growth factor (TGF)-beta. Given the inhibitory activity of TGF-beta, its elevated levels may contribute to the selective pressure of Campath-1H, leading to the emergence of PNH-like lymphocyte